Cyclin-dependent kinases (CDKs) are the fundamental regulators of the cell cycle in eukaryotes, which are activated by binding to cyclins whose abundance changes during the cell cycle. A single cyclin and multiple CDKs function in yeast cell cycle control, whereas multiple cyclins and multiple CDKs control the cell cycle progression in animals. Trypanosomes appear to employ a cell cycle control system that is strikingly different from that in fungi and animals. A PHO80-like cyclin, CYC2, and a CDK-related kinase, CRK1, control the G1/S transition, whereas a B-type cyclin, CYC6, and another CDK-related kinase, CRK3, govern the G2/M transition. However, how CRK1 exert their roles in cell cycle progression remains elusive, mainly because its downstream targets are not known. The current proposal aims to understand the mechanisms underlying the G1/S cell cycle transition and the molecular basis for the distinct cell cycle regulation between different life cycle forms of T. brucei. In aim 1 of this proposal, we plan to apply chemical genetic approach to identify CRK1 substrates from the procyclic and bloodstream forms and to comparatively analyze the CRK1-regulated cellular pathways between the two forms. Aim 2 is to understand the mechanisms underlying the cell cycle-dependent protein translation initiation, with a focus on the regulation of two translation initiation factors by CRK1. Aim 3 is to dissect the role of CRK1 in DNA replication initiation by investigating CRK1-mediated regulation of the DNA replication factors in the Cdc45-Mcm2-7-GINS complex. Through molecular, cell biological, chemical genetic, and biochemical approaches, our overall goal in this proposal is to understand the mechanistic roles of CRK1 in the G1/S control through regulating the DNA replicative helicase and in G1-specific protein translation initiation through regulating translation initiation factors. The outcomes from these studies not only will significantly advance our understanding of the mechanisms of cell cycle transitions in trypanosomes, but also could validate CRK1 and its trypanosome-specific downstream pathways as drug targets for anti-trypanosome chemotherapy.